Differentiated 3T3-L1 adipocytes were used to study the control and metabolic role of cellular phosphodiesterases. Incubation of these cells with 100 pM insulin or 1 MuM isoproterenol resulted in activation of the particulate cAMP phosphodiesterase within 10 min. The effect of insulin but not that of isoproterenol was prevented by prior treatment of cells with pertussis toxin. In cells maintained for 2 weeks in hypothyroid medium (medium with 10% serum from a hypothyroid calf), basal lipolysis and cAMP levels and sensitivity to isoproterenol stimulation were decreased. Opposite changes in lipolysis and cAMP levels occurred in cells maintained in hyperthyroid medium. In hypothyroid cells, particulate and soluble cAMP phosphodiesterase activity was increased. Sensitivity of adenyl cyclase in these cells to stimulation by isoproterenol was decreased, but there was no change in Beta-adrenergic receptor number or affinity. Conversely, in hyperthyroid cells, particulate and soluble cAMP phosphodiesterase were decreased; adenyl cyclase activity and sensitivity to stimulation by isoproterenol were increased, but there was no change in Beta-receptor number or affinity. The change in hormone-stimulated lipolysis observed in different thyroid states in these cells may be secondary to changes in both phosphodiesterase activity and receptor-cyclase coupling. The soluble cAMP phosphodiesterase activity in these cells is more sensitive to inhibition by Ro 20-1724, but the particulate activity is more sensitive to inhibition by cilostamide. In cells incubated with cilostamide, stimulated cAMP levels were lower, but lipolysis (basal and isoproterenol sensitivity) was greater than in cells incubated with Ro 20-1724. These findings suggest that the particulate cAMP phosphodiesterase may be important in the metabolism of cAMP involved in the regulation of lipolysis.